Streptococcus oralis is among the earliest colonizers of the tooth surface during plaque formation. As such, its enzymatic activities may influence ecologic succession on the tooth surface. In the current study, we used zymograms and preparative polyacrylamide gel electrophoresis to identify and purify a protease from S. oralis (sanguis) C104. Proteases from 5. oralis C104 were detected in cell pellets at 133, 146 and 176 kDa as clear proteolytic bands on gelatin‐substrate zymograms. Preparations of the major (146 kDa) protease were obtained by continuous‐elution electrophoresis. The protease was active over the pH range of 7 to 9 with optimum activity between pH 8 and 9. Protease activity was inhibited by several serine protease inhibitors including phenylmethylsulfonyl fluoride, di‐iso‐propyl‐phosphofluoridate and aprotinin. The protease showed highest hydrolytic activity against azoalbumin and Bz‐Pro‐Phe‐Arg‐NA. Immunofluorescence studies with a polyclonal antiserum to the 146–kDa protease suggest it is present on the cell surface of S. oralis C104. Zymograms of cell pellets from other S. oralis strains as well as S. sanguis and Streptococcus mitis suggest that functionally similar proteases are elaborated by many early colonizers of the tooth surface.